ABSTRACT

Mycobacterium tuberculosis must sense and adapt to host environmental cues to establish and maintain an infection. The two-component regulatory system PhoPR plays a central role in sensing and responding to acidic pH within the macrophage and is required for M. tuberculosis intracellular replication and growth in vivo. Therefore, the isolation of compounds that inhibit PhoPR-dependent adaptation may identify new antivirulence therapies to treat tuberculosis. Here, we report that the carbonic anhydrase inhibitor ethoxzolamide inhibits the PhoPR regulon and reduces pathogen virulence. We show that treatment of M. tuberculosis with ethoxzolamide recapitulates phoPR mutant phenotypes, including downregulation of the core PhoPR regulon, altered accumulation of virulence-associated lipids, and inhibition of Esx-1 protein secretion. Quantitative single-cell imaging of a PhoPR-dependent fluorescent reporter strain demonstrates that ethoxzolamide inhibits PhoPR-regulated genes in infected macrophages and mouse lungs. Moreover, ethoxzolamide reduces M. tuberculosis growth in both macrophages and infected mice. Ethoxzolamide inhibits M. tuberculosis carbonic anhydrase activity, supporting a previously unrecognized link between carbonic anhydrase activity and PhoPR signaling. We propose that ethoxzolamide may be pursued as a new class of antivirulence therapy that functions by modulating expression of the PhoPR regulon and Esx-1-dependent virulence.

INTRODUCTION

Mycobacterium tuberculosis is a successful pathogen because it overcomes a variety of obstacles raised by the host immune response. The ability of M. tuberculosis to sense host immune pressures and orchestrate adaptive responses to these cues is essential for pathogen virulence. It follows that the identification of chemical compounds that disrupt the ability of M. tuberculosis to sense and respond to host cues may function to attenuate pathogen virulence.

M. tuberculosis uses environmental pH as a cue to modulate its physiology. These pH-dependent adaptations play a central role in M. tuberculosis pathogenesis (1). Transcriptional profiling of M. tuberculosis-infected macrophages identified an M. tuberculosis regulon induced at acidic pH (2) that significantly overlaps the PhoPR two-component regulatory system regulon (3–5), suggesting a role for PhoPR in pH-driven adaptation. phoPR-inactivated M. tuberculosis mutant strains are highly attenuated during mouse and guinea pig infections, supporting that this pathway is a suitable target for new drug development (6, 7).

Conventional antimicrobial discovery campaigns seeking to identify bactericidal or bacteriostatic compounds are often performed in vitro. However, many targets required for M. tuberculosis pathogenesis, including environmental sensing pathways, are essential only in vivo and would be missed using standard approaches. Compounds that target in vivo essential bacterial virulence factors are known as antivirulence therapies (8). Antivirulence therapies are advantageous over traditional antibiotics because they preserve endogenous microbiota by targeting pathogen-specific pathways and may reduce selective pressure for the development of resistance, since bacterial growth is not directly targeted. Several antivirulence compounds that target bacterial virulence regulators, quorum sensing, toxin production and delivery, and adhesion and colonization within the host have been discovered (8–13). The goal of this study was to identify compounds that inhibit the M. tuberculosis PhoPR regulon and may thus function as antivirulence therapies.

MATERIALS AND METHODS

Bacterial strains and growth conditions.M. tuberculosis experiments, unless otherwise stated, were performed with M. tuberculosis strain CDC1551. The CDC1551(aprA′::GFP) fluorescent reporter strain was generated by fusing the promoter region of aprA (Mcr7 gene, Rv2395a) upstream of green fluorescent protein (GFP) in a replicating plasmid and transforming the plasmid into CDC1551 (5). The phoP::Tn and ΔphoPR mutants were previously described (5, 14). Cultures were maintained in standing tissue culture flasks in 7H9 Middlebrook medium supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) and 0.05% Tween 80 and incubated at 37°C with 5% CO2.

High-throughput screening assay and data analysis.An ∼220,000-compound library arrayed in 384-well optical microtiter plates was provided by the Institute of Chemistry and Cell Biology-Longwood/National Small Molecule Screening and Medicinal Chemistry Core (ICCB-Longwood/NSRB) at Harvard University. Two columns of each plate were left blank for positive and negative controls of 0.3 μM rifampin and dimethyl sulfoxide (DMSO), respectively. M. tuberculosis CDC1551(aprA′::GFP) was grown in Middlebrook 7H9 medium and buffered to pH 7.0 with 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS) to mid- to late-log phase. The cultures were then pelleted, resuspended in 7H9 buffered to pH 5.7 with 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), and dispensed into the 384-well assay plates at an optical density (OD) of 0.2. The plates were incubated for 6 days at 37°C, and both fluorescence and OD were measured on a plate reader. For analysis of the hits, fluorescence and growth inhibition were normalized based on the rifampin (100%) and DMSO (0%) controls. Potential PhoPR regulon inhibitors were flagged as compounds that had (i) >35% fluorescence inhibition and (ii) ≥1.5-fold greater fluorescence inhibition than growth inhibition.

For determination of 50% effective concentrations (EC50s), M. tuberculosis CDC1551(aprA′::GFP) was grown to mid- to late-log phase in noninducing medium (7H9 [pH 7.0]), pelleted, and resuspended in GFP-inducing medium (7H9 [pH 5.7]) in the presence of a 14-point dilution series (2.5-fold) of ethoxzolamide (ETZ), at a final concentration ranging from 3 nM to 500 μM. Fluorescence and growth inhibition were normalized based on the rifampin (100%) and DMSO (0%) controls. EC50s were calculated based on a variable-slope four-parameter nonlinear least-squares regression model in the GraphPad Prism software package (version 6).

Transcriptional profiling and data analysis.For high-throughput RNA sequencing (RNA-seq) experiments, M. tuberculosis cultures were grown at 37°C in T-25 vented standing tissue culture flasks in 8 ml of buffered 7H9 medium seeded at an initial OD of 0.2. Three conditions were examined with two biological replicates: (i) DMSO treatment at pH 7.0, (ii) DMSO treatment at pH 5.7, and (iii) 40 μM ETZ treatment at pH 5.7. The phoP::Tn mutant was grown in a similar manner and treated with DMSO at pH 5.7. Following 6 days of incubation, total bacterial RNA was extracted as previously described (2). RNA-sequencing, data analysis, and quantitative PCR (qPCR) were performed as described by Baker, Johnson, and Abramovitch (15), with minor modifications (see the supplemental material). Differentially expressed genes were determined to be statistically significant based on a false-discovery rate adjusted P value of <0.05.

Carbonic anhydrase activity assay.Carbonic anhydrase (CA) activity was measured using a modified Maren endpoint method (17) based on acidification of the medium by CA conversion of CO2. WT M. tuberculosis CDC1551 cultures were grown for 6 days at 37°C in T-150 vented standing tissue culture flasks in 50 ml of 7H9 medium seeded at an initial OD of 0.2. Two conditions were examined: (i) DMSO treatment at pH 5.7 and (ii) ETZ treatment (80 μM) at pH 5.7. Cells were then were pelleted, washed once in cold assay buffer (20 mM Tris, 20 mM NaCl [pH 8.3]), resuspended in 500 μl of cold assay buffer, and transferred to 2-ml screw-cap tubes containing 200 μl of 0.1-mm glass beads. Cell lysis was achieved by bead beating at maximum speed for 2 min. Lysates were clarified by centrifugation at 21,000 × g for 1 min. The clarified lysates were transferred to new tubes and kept on ice. The CA assay apparatus utilized 15- by 88-mm Pyrex glass tubes, 14-mm serum stoppers, 18-gauge 1.5-in. needles, 1-ml syringes, 1-mm tubing, sample evaporator needles, and 0.22-μm syringe filters (to prevent aerosol escape). All assay components were kept on ice before and during the assay. Four hundred fifty microliters of double-distilled water (ddH2O) was added to the tube with 50 μl of cell lysate. CO2 was bubbled in at a constant rate, 500 μl of cold color indicator buffer (20 mM imidazole, 5 mM Tris, 0.2 mM 4-nitrophenol) was added, and timing was initiated. The color was monitored until clearing, comparing it to a previously cleared sample. The data are representative of two biological replicates, with similar findings in each experiment.

Cytoplasmic pH and phagosome acidification measurement.Cytoplasmic pH was measured as previously described by Purdy, Niederweis, and Russell (18). WT M. tuberculosis CDC1551 was grown in a similar manner as in the transcriptional profiling experiments. Four conditions were examined: (i) DMSO treatment at pH 7.0, (ii) DMSO treatment at pH 5.7, (iii) 80 μM ETZ treatment at pH 7.0, and (iv) 80 μM ETZ treatment at pH 5.7. Cytoplasmic pH was measured following 6 days of incubation. Phagosome acidification was measured using pHrodo-labeled beads (Life Technologies) fed to BMDMs in a 96-well black assay plate. One column did not contain any cells for a medium-and-beads-only control. Five conditions were examined: (i) 100 μM ETZ treatment, (ii) 80 μM ETZ treatment, (iii) 40 μM ETZ treatment, (iv) 100 nM concanamycin A (CcA), and (v) an equivalent volume of DMSO. Cells were pretreated for 4 h with ETZ or DMSO before the addition of particles. CcA was added 30 min prior to assay initiation. pHrodo green-labeled particles (excitation [ex.], 509 nm; emission [em.], 533 nm) were added to each well in prewarmed live cell imaging solution (Life Technologies) at 1 mg/ml. The assay was carried out in a PerkinElmer plate reader at 37°C for 100 min, taking readings at 5-min intervals.

Analysis of mycobacterial lipids.Lipid analysis was conducted as previously described (15). Briefly, M. tuberculosis cultures were grown at 37°C in T-75 vented standing tissue culture flasks in 40 ml of minimal medium, supplemented with 10 mM pyruvate, and seeded at an initial OD of 0.1. Four conditions were examined: (i) DMSO treatment at pH 7.0, (ii) DMSO treatment at pH 5.7, (iii) 80 μM ETZ treatment at pH 7.0, and (iv) 80 μM ETZ treatment at pH 5.7. Following 3 days of incubation, lipids were radiolabeled by adding 8 μCi of [1,2-14C]sodium acetate to each culture. Following 6 days of labeling, the lipids were extracted and analyzed by thin-layer chromatography (TLC). Total extractable lipid 14C incorporation was determined using a scintillation counter. To analyze lipid species, 20,000 cpm was loaded at the origin of a 100-cm2 high-performance TLC silica gel 60 aluminum sheet. To separate sulfolipid (SL) and triacylglycerol (TAG), TLC plates were developed with chloroform-methanol-water (90:10:1 [vol/vol/vol]) and hexane-diethyl ether-acetic acid (80:20:1 [vol/vol/vol]) solvent systems, respectively (15). To examine phthiocerol dimycocerosate (PDIM) accumulation, the two-dimensional (2D) TLC plate was developed with (i) petroleum ether-ethyl acetate (98:2 [vol/vol]) and (ii) petroleum ether-acetone (98:2 [vol/vol]) (5). Radiolabeled lipids were detected with a phosphor screen and Typhoon imager and quantified using the ImageJ software (19). Radiolabeling experiments, lipid extractions, and analyses were repeated in two independent biological replicates, with similar findings in the two replicates. TAG and PDIM species were previously identified using these solvent systems and mass spectrometry (5). SL identity was confirmed using an SL standard (provided by BEI Resources) that was visualized by staining with phosphomolybdic acid, followed by charring (see Fig. S3C in the supplemental material).

Analysis of Esx-1 protein export.M. tuberculosis Erdman and Δesat-6 strains were a gift from Jeffery S. Cox (20). M. tuberculosis was cultured in 7H9 medium, collected by centrifugation, washed once with 5 ml of Sauton's broth, and resuspended in 50 ml of Sauton's broth in the presence or absence of 80 μM ETZ. After 5 days of growth at 37°C, the cells were diluted, washed, and treated as described above. The cultures were grown at 37°C with gentle shaking for 4 days. Lysates were prepared as described previously (20). The secreted protein fraction was concentrated using Amicon filter systems with a 3-kDa molecular mass cutoff, and total protein concentrations were determined by the Micro BCA assay (Promega). Before being transferred to nitrocellulose, 13.5 μg of cell lysates and culture filtrates were separated on a 4 to 20% precast gel (Bio-Rad). The nitrocellulose membranes were incubated with antibodies against the 6-kDa early secretory antigenic target (ESAT-6) (catalog no. ab26246; Abcam), the 10-kDa culture filtrate protein (CFP-10), M. tuberculosis protein 32 (Mpt32), or RNA polymerase beta (RNAP-β) (catalog no. 8RB13; Abcam) and detected using chemiluminescence (LumiGLO) (21). The following reagents were obtained through BEI Resources, National Institute of Allergies and Infectious Diseases (NIAID), NIH: polyclonal anti-M. tuberculosis CFP-10 (gene Rv3874) (antiserum, rabbit), NR-13801; polyclonal anti-M. tuberculosis Mpt32 (gene Rv1860) (antiserum, rabbit), NR-13807.

Macrophage infections.Macrophage infections were performed as described previously (16). Briefly, BMDMs were infected at a multiplicity of infection (MOI) of 1:1 with M. tuberculosis CDC1551 in 24-well tissue culture-treated plates. Infected BMDMs were treated with 80 μM ETZ or an equivalent volume of DMSO every 2 days for 9 days total. At days 3, 6, and 9, intracellular bacteria were quantified by lysing macrophage monolayers and performing serial dilution plating of lysates on 7H10 agar. Each treatment was added to three separate wells per plate to provide three biological replicates per time point. Survival assays were repeated in three independent experiments, with similar findings in each experiment. For fluorescence microscopy experiments, macrophages were seeded on glass coverslips before infection with M. tuberculosis CDC1551(aprA′::GFP smyc′::mCherry). Samples were treated every 2 days with 100 μM ETZ or an equal volume of DMSO for 9 days. Monolayers were then fixed in 4% paraformaldehyde and imaged by confocal microscopy. Imaging experiments were repeated with three biological replicates, with similar results.

Quantitative single-cell imaging of M. tuberculosis exposure to ETZ in mice.C57BL/6 mice were infected via the intranasal route with 1,000 CFU of the Erdman (aprA′::GFP smyc′::mCherry) dual fluorescent reporter. Mice were treated with 100 mg/kg of body weight of ETZ or an equal volume of 0.25% carboxymethyl cellulose (CMC) 5 days per week for 4 weeks via oral gavage. After 4 weeks, the right lung was homogenized and plated for enumeration of the bacterial load, and the left lung was fixed in 4% paraformaldehyde before being transferred to 30% sucrose for confocal microscopy imaging and evaluation for histopathological hallmarks of tuberculosis (TB) disease (see the supplemental material). This experiment was repeated, and the data from the two independent biological replicates were similar and therefore combined. Mice were euthanized by carbon dioxide asphyxiation, followed by cervical dislocation. The animal experiments were conducted in accordance with protocols approved by the Michigan State University Institutional Animal Care and Use Committee.

RESULTS

Identification of ethoxzolamide as an inhibitor of the PhoPR regulon.We employed a whole-cell phenotypic screen to identify inhibitors of the PhoPR regulon. The CDC1551(aprA′::GFP) fluorescent reporter exhibits pH-inducible fluorescence that is fully dependent on PhoPR (5). Although PhoPR is required for growth in vivo, a PhoPR mutant grows well in rich medium at acidic pH; therefore, compounds that inhibit pH-inducible reporter fluorescence, but not growth, are anticipated to be inhibitors of the PhoPR pathway. To discover inhibitors of the PhoPR regulon, we conducted a high-throughput screen (HTS) of an ∼220,000-compound library composed of small molecules representing broad chemical diversity. The reporter strain was grown in 384-well plates containing the compound library (at a concentration of ∼10 μM) in rich medium buffered to pH 5.7. Following 6 days of incubation, plates were examined for GFP fluorescence and growth. Ethoxzolamide (ETZ) (Fig. 1A) was identified as a compound that inhibits M. tuberculosis reporter fluorescence but does not reduce growth. ETZ inhibits CDC1551(aprA′::GFP) reporter fluorescence with a half-maximal effective concentration (EC50) of 5.6 μM at pH 5.7 (Fig. 1B). Growth was mostly unaffected at pH 5.7 across the range of ETZ concentrations tested (Fig. 1B). Basal aprA expression is also dependent on PhoPR at neutral pH, and we observed inhibition of reporter fluorescence at pH 7.0, with a similar EC50 of 4.7 μM (see Fig. S1A in the supplemental material). ETZ is a carbonic anhydrase (CA) inhibitor that has been shown to inhibit recombinant M. tuberculosis CA proteins (22). We examined if ETZ inhibits M. tuberculosis CA activity and observed full inhibition of CA activity in M. tuberculosis whole cells treated with ETZ for 6 days (Fig. 1C). ETZ did not quench GFP fluorescence or alter the pH of the culture medium (see Fig. S1B in the supplemental material), and it caused only a slight acidification of cytoplasmic pH (Fig. 1D). These data support two novel findings: (i) ETZ inhibits the PhoPR regulon, and (ii) a previously unrecognized link may exist between M. tuberculosis CA activity and PhoPR signaling.

Ethoxzolamide inhibits M. tuberculosis CDC1551(aprA′::GFP) fluorescence and M. tuberculosis carbonic anhydrase activity but does not alter cytoplasmic pH. (A) Chemical structure of ethoxzolamide (ETZ) (6-ethoxy-1,3-benzothiazole-2-sulfonamide). (B) ETZ inhibits PhoPR-dependent CDC1551(aprA′::GFP) fluorescence in a concentration-dependent manner at pH 5.7, with an EC50 of 5.6 μM and little effect on growth. (C) M. tuberculosis carbonic anhydrase activity is not detectable (n.d.) in whole cells when treated with ETZ (80 μM) compared to a DMSO control. (D) M. tuberculosis treated with ETZ (80 μM) for 6 days exhibits no change in cytoplasmic pH at pH 7.0 and only slightly acidified cytoplasm (<0.1 pH units) at pH 5.7. The data are representative of at least two biological replicates, and the error bars are the standard deviations from at least three technical replicates. *, P < 0.05, calculated based on a two-tailed t test.

Prior transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) studies have defined a core regulon that is directly regulated by PhoP and induced by acidic pH (2–5, 23, 24). To determine if ETZ inhibits the PhoPR regulon, we performed RNA-seq transcriptional profiling. M. tuberculosis CDC1551 was inoculated into rich medium buffered to pH 5.7 in the presence of either 40 μM ETZ or an equivalent volume of DMSO. As a positive control for PhoPR-regulated genes, a DMSO-treated phoP transposon mutant strain (phoP::Tn) was cultured under the above-described conditions. To identify genes regulated by acidic pH, we also examined the transcriptional profile of DMSO-treated M. tuberculosis at pH 5.7 compared to that at pH 7.0. After 6 days of incubation, total RNA was isolated and subjected to RNA sequencing. ETZ treatment of M. tuberculosis caused the downregulation (>2-fold, P < 0.05) of 45 genes (Fig. 2; see also Table S2B in the supplemental material). Remarkably, all 45 of these genes were also downregulated in the phoP::Tn mutant, and 40 were induced by acidic pH (Fig. 2B and C; see also Tables S1A and S3B in the supplemental material). ETZ-downregulated genes include many genes that were previously shown to be directly controlled by PhoP and that are involved in lipid synthesis, carbon metabolism, and virulence (Fig. 2C) (23, 24). Many of the remaining 137 genes downregulated in the phoP::Tn mutant but not by ETZ (>2-fold, P < 0.05) are significantly downregulated by ETZ but <2-fold (e.g., espR, espA, and esxA) (Fig. 2A; see also Table S5 in the supplemental material). To confirm the RNA-seq results, we conducted semiquantitative real-time PCR on the phoP gene and two well-characterized PhoP-regulated genes, aprA and pks2. In ETZ-treated M. tuberculosis, we observed that aprA and pks2 were downregulated >5-fold at both pH 7.0 and 5.7 (see Fig. S2 in the supplemental material). phoP did not exhibit substantial differential regulation by ETZ, demonstrating that ETZ does not act by modulating phoP gene expression. These findings validate ETZ as an inhibitor of the core PhoPR regulon.

Lipid synthesis and Esx-1 protein secretion are modulated by ethoxzolamide.PhoPR controls cell envelope lipids and Esx-1-dependent secretion of ESAT-6 (EsxA) (25, 26). Therefore, we examined if ETZ modulates these virulence factors. The PhoPR-dependent mmpL8-pks2 operon (genes Rv3823c to Rv3825c) is responsible for the production of sulfolipid (SL) and is strongly induced at acidic pH (3, 15, 27, 28). Transcriptional profiling data demonstrated an ∼5-fold downregulation of this operon when treated with ETZ at acidic pH (Fig. 2C), suggesting that, similarly to knockout of phoPR (ΔphoPR mutant), ETZ may downregulate the accumulation of SL. To test this hypothesis, 14C-radiolabeled lipids were isolated from wild-type (WT) and ΔphoPR mutant strains grown at pH 7.0 and pH 5.7 treated with either ETZ or an equal volume of DMSO. A lipid migrating with a position consistent with SL was induced ∼2.5-fold at pH 5.7 compared to pH 7.0 in DMSO, whereas this lipid was not detected in ETZ-treated cells and the ΔphoPR mutant (Fig. 3A; see also Fig. S3A to C in the supplemental material). It has been shown that mutant strains with reduced accumulation of PhoPR-regulated lipids compensate by overaccumulating other long-chain fatty acids, such as triacylglycerol (TAG) and phthiocerol dimycocerosate (PDIM) (5, 15, 29). At pH 5.7, compared to the DMSO control, TAG was induced 5.5-fold and 6.5-fold in ETZ-treated M. tuberculosis and the ΔphoPR mutant strain, respectively (Fig. 3B; see also Fig. S3A in the supplemental material). Similarly, at pH 5.7, we observed a 2-fold increase in the accumulation of PDIM species in the ETZ-treated samples compared to the DMSO-treated cells (Fig. 3C). Therefore, ETZ treatment recapitulates the phenotype of the ΔphoPR mutant strain for alteration of lipid species production.

ETZ reduces the accumulation of sulfolipid and enhances accumulation of triacylglycerol and phthiocerol dimycocerosate. Radio-TLC showing that M. tuberculosis treated with 80 μM ETZ and the ΔphoPR mutant strain exhibit a lack of accumulation of sulfolipid (SL) (denoted by *) (A) and enhanced accumulation of triacylglycerol (TAG) (denoted by *) (B). (C) 2D radio-TLC demonstrating that ETZ treatment increases the accumulation of TAG (spot 1), phthiocerol dimycocerosate (PDIM) species (spots 2 and 3), and an unidentified lipid species (spot 4) at pH 5.7 compared to a DMSO control. The data are representative of two biological replicates, with similar findings in the two experiments.

The ESAT-6 protein is expressed in a phoP mutant strain, but it is not exported from the bacterial cell (25). Therefore, we tested if ETZ treatment altered the secretion of Esx-1-exported proteins. M. tuberculosis Erdman was grown in rich medium and passaged twice in Sauton's minimal medium to measure Esx-1 export. During growth in Sauton's medium, the cells were untreated, treated with ETZ during both passages (WT++, Fig. 4), or treated with ETZ in the final passage only (WT−+, Fig. 4). ETZ did not affect ESAT-6 and CFP-10 expression but strongly and selectively reduced their secretion, as it did not alter that of the Sec-secreted protein Mpt32 (Fig. 4). Moreover, transcriptional profiling revealed that espR and the espACD operon were significantly downregulated (>1.5-fold, P < 0.05) in ETZ-treated cultures (see Table S5 in the supplemental material). Recently, Cao and colleagues have shown that PhoPR directly regulates EspR-dependent expression of espACD (26). EspA secretion and ESAT-6 secretion are mutually dependent, and the loss of espR and espA expression leads to reduced Esx-1 function, loss of ESAT-6 secretion, and attenuated virulence (23, 30). Therefore, both transcriptional profiling in M. tuberculosis CDC1551 and biochemical approaches in M. tuberculosis Erdman support that ETZ inhibits Esx-1 secretion. Selective inhibition of PhoPR-dependent physiologies, such as SL synthesis and Esx-1 export, is predicted to reduce M. tuberculosis virulence (31).

ETZ inhibits Esx-1 protein export. Western blot analysis of cell lysates (CL) and culture filtrates (CF) of wild-type (WT) M. tuberculosis Erdman and Δesat-6 strains grown in Sauton's medium with or without addition of ETZ (80 μM). The RNAP-β subunit served as a control for lysis and as a loading control for CL, and Mpt32 served as loading control for CF and as a measure of Sec secretion. The CFP-10 and ESAT-6 antibodies detected the EsxB and EsxA proteins, respectively, from the WT M. tuberculosis Erdman strain. ETZ treatment inhibits the secretion of ESAT-6 and CFP-10. −−, no ETZ added; ++, ETZ added in both passages; −+, ETZ added in second passage only. The data are representative of three biological replicates.

Ethoxzolamide inhibits M. tuberculosis PhoPR regulon expression and growth in infected macrophages and mice.The PhoPR regulon is induced within 20 min of M. tuberculosis phagocytosis by macrophages and remains induced over a period of ≥14 days (32). To determine if ETZ modulates the PhoPR regulon in macrophages, we infected murine bone marrow-derived macrophages (BMDMs) with the CDC1551(aprA′::GFP smyc′::mCherry) reporter strain. This strain exhibits PhoPR-inducible expression of GFP and constitutive expression of mCherry (5). The infected macrophages were treated with ETZ or DMSO, and single-cell reporter fluorescence was quantified at 6 days postinfection. ETZ treatment caused >90% inhibition of reporter GFP fluorescence in infected macrophages (Fig. 5A and B). Moreover, in a 9-day macrophage survival assay, ETZ treatment significantly inhibited the ability of M. tuberculosis to grow intracellularly (Fig. 5C). Notably, ETZ-treated and -untreated bacteria exhibited similar initial decreases in growth during the first 3 days of infection. However, the untreated M. tuberculosis successfully adapted to the macrophage environment and grew ∼0.5 log over the next 6 days, while the ETZ-treated cells could not transition to a growth phase. This phenotype is similar to that observed with a PhoPR mutant strain (6) and is consistent with ETZ functioning as an antivirulence agent targeting PhoPR-dependent macrophage adaptation.

ETZ reduces PhoPR-regulated gene expression and survival in macrophages. Primary murine BMDMs were infected with the M. tuberculosis CDC1551(aprA′::GFP smyc′::mCherry) reporter at an MOI of 1:1 and treated with DMSO or ETZ (100 μM) every 2 days for 9 days. (A) Confocal microscopy images demonstrate that ETZ treatment inhibits the PhoPR regulon in infected macrophages. The merged images show GFP (PhoPR-inducible signal) and mCherry (constitutive signal) fluorescence. (B) Single-cell quantification of reporter fluorescence shows that ETZ significantly downregulates PhoPR-dependent GFP fluorescence compared to a DMSO control. Statistical significance was calculated based on the Mann-Whitney rank test (*, P < 0.001). (C) Treatment of infected BMDMs with 80 μM ETZ reduces growth ∼1 log compared to the DMSO control. The data shown are three biological replicates and representative of three independent experiments. Statistical significance was calculated based on a two-tailed t test (*, P < 0.001). The error bars are the standard deviations from three biological replicates.

ETZ is also a eukaryotic CA inhibitor. Therefore, it is possible that the observed intracellular M. tuberculosis phenotypes may, in part, be driven by ETZ targeting macrophage CA activity. Because PhoPR is induced by acidic pH in macrophages, we examined if ETZ inhibits phagosome acidification. BMDMs were treated with DMSO, ETZ, or concanamycin A (an inhibitor of the vacuolar ATPase) 4 h prior to being fed with beads coated with the pH-sensitive dye pHrodo. Phagosome acidification was monitored for 100 min using a plate reader. Even at concentrations as high as 100 μM, ETZ did not alter phagosome acidification (see Fig. S1C in the supplemental material), supporting the idea that ETZ-induced inhibition of M. tuberculosis growth is mediated by PhoPR regulon inhibition rather than by altering the environment within the macrophage phagosome.

We next examined if ETZ can modulate the PhoPR regulon in vivo. C57BL/6 mice were infected with the Erdman (aprA′::GFP smyc′::mCherry) dual fluorescent reporter strain, and the mice were treated orally with either 100 mg/kg of ETZ or an equal volume of 0.25% carboxymethyl cellulose (as a mock treatment) 5 days per week for 4 weeks. In animals, ETZ was previously reported to have a half-life of 2.5 to 5.5 h, is rapidly absorbed, and has a 50% lethal dose (LD50) of ∼1,000 mg/kg, with no observable drug-related organ lesions at 100 mg/kg (33–38). Quantitative single-cell imaging was used to measure the induction of reporter fluorescence in lung tissues. For this approach, lung tissue was sectioned and imaged by confocal microscopy, and bacterial fluorescence was quantified using the Volocity image analysis software (14, 39). Individual bacterial cells were identified using the constitutive mCherry signal, and then GFP fluorescence was measured for ∼3,000 bacterial cells across multiple fields of view from lungs of mice in the same treatment group. Two independent experiments were conducted with similar results; therefore, the data from the two experiments were combined for analysis. The Erdman (aprA′::GFP smyc′::mCherry) reporter exhibited a strong induction of GFP fluorescence in the lungs of mock-treated, infected mice (Fig. 6A). Notably, single cells in the mock-treated samples exhibited substantial heterogeneity of GFP fluorescence, providing additional evidence that M. tuberculosis experiences a variety of microenvironments during the course of infection (Fig. 6B). In contrast, ETZ strongly downregulated GFP reporter fluorescence in mouse lungs, with 3-fold inhibition of GFP signal compared to that in the mock-treated control (Fig. 6B). Importantly, the distribution of reporter fluorescence was dramatically altered by ETZ treatment, with a more homogeneous population of cells expressing low levels of GFP. For example, 49% of M. tuberculosis cells in ETZ-treated lungs quantified for GFP fluorescence are in the bottom decile of fluorescence (<1,000 GFP/μm2), compared to 5% of cells in the mock-treated mice (Fig. 6B). This approach demonstrates that quantitative analysis of fluorescent M. tuberculosis reporter strains in host tissues can be applied as a biomarker for pathway-specific drug exposure in vivo. ETZ-treated mouse lungs did not have a significant reduction in the area of the lung showing M. tuberculosis-associated granulomatous pneumonia (see Fig. S4 in the supplemental material). The lesions in both mock- and ETZ-treated mice consisted of multifocal infiltrates of epithelioid to multinucleated macrophages, obliterating and expanding alveolar lumina, often with cuffs of lymphocytic infiltrate surrounding adjacent arterioles (see Fig. S5 in the supplemental material). There were occasional instances of lymphohistiocytic pleuritis in both groups. The effect of ETZ treatment on bacterial survival in vivo was also examined. We observed a significant 0.72-log reduction of bacterial survival in the lungs of ETZ-treated mice compared to the mock-treated control (Fig. 6C). Therefore, adequate ETZ exposure was achieved in mouse lungs to repress the PhoPR regulon and attenuate virulence. Together, these data support that ETZ functions by an antivirulence mechanism and attenuates M. tuberculosis virulence in vivo by (i) targeting M. tuberculosis inside macrophages and the mouse lung, (ii) downregulating the PhoPR regulon, and (iii) reducing the expression of virulence pathways, such as lipid metabolism and Esx-1 secretion.

DISCUSSION

We identified ETZ as an inhibitor of the PhoPR regulon by conducting a whole-cell HTS using an acidic-pH-inducible PhoPR-dependent reporter strain. This HTS is particularly unique in that it was performed in vitro but identified an inhibitor of a target, the PhoPR regulon, which is required for growth only in macrophages and in vivo. To our knowledge, there are no known chemical inhibitors of the PhoPR regulon. Recently, Rybniker and colleagues identified an antivirulence inhibitor of Esx-1 secretion using an anticytolytic screen (31), although the mechanism of this compound appears to be PhoPR independent. ETZ does not alter M. tuberculosis cytoplasmic pH homeostasis (Fig. 1D) and is thus acting by a mechanism that is distinct from compounds found to modulate cytoplasmic pH homeostasis (40–42). ETZ therefore represents a first-in-class agent and strategy to inhibit M. tuberculosis pH-dependent adaptation and virulence. Inhibiting an adaptation pathway required for pathogen virulence in vivo exemplifies an antivirulence strategy, a new approach to drug development that is proposed to slow the evolution of drug resistance (8–12).

ETZ is an FDA-approved drug prescribed for the treatment of glaucoma, and it is utilized as a general diuretic through the CA inhibitory properties of the compound (43). Because ETZ is already proven to be safe in humans, it may be possible to repurpose ETZ for the treatment of M. tuberculosis. The PhoPR mutant strain is highly attenuated in animal infections and is currently being developed as a potential attenuated M. tuberculosis vaccine (44). Thus, it will be of considerable interest to continue the development of ETZ for the treatment of TB, possibly as an adjunct therapy with current drug regimens. Given the incomplete inhibition of the PhoPR regulon in vivo (Fig. 6B), improvements to compound potency or lung distribution may improve ETZ efficacy in vivo.

The mechanism by which ETZ modulates the PhoPR regulon remains to be determined. It is possible that ETZ functions by directly targeting PhoPR or targeting a PhoPR-associated regulator (such as WhiB3 [45]); however, given that ETZ has been shown to inhibit M. tuberculosis CA activity in whole cells (Fig. 1C), we hypothesize that ETZ may indirectly inhibit the PhoPR regulon by interfering with CA activity (Fig. 7). M. tuberculosis carries 3 CA genes, Rv3273, Rv1284, and Rv3588c, of which Rv1284 and Rv3588c are required for survival in mice (46). Because CA catalyzes the reaction of CO2 to bicarbonate and a proton, CA may function to modulate local pH (47, 48). We did not, however, observe any change in the pH of the culture medium and only a slight change of <0.1 pH units in the M. tuberculosis cytoplasm, suggesting that if the mechanism is pH dependent, it may be localized to the cell envelope. Depending on the cellular localization of CAs (predicted to be extracytoplasmic [49]) and anion/cation homeostasis (e.g., protons, bicarbonate, ammonium, and chloride), CA activity could potentially promote an acidified or alkalinized cell envelope (50). Given that ETZ inhibits PhoPR signaling and PhoPR is induced by acidic pH, we predict that CA may promote an acidified pseudoperiplasm. In this model (Fig. 7), CA generates extracellular proton and bicarbonate ions. The bicarbonate can be transported by a bicarbonate transporter (BT) (M. tuberculosis has several predicted BT genes) to maintain the cytoplasmic pH. CA will cause protons to accumulate in the pseudoperiplasm, causing the activation of the PhoR kinase and the PhoPR pathway. ETZ would inhibit this process by inhibiting CA and reducing the accumulation of protons in the pseudoperiplasm (Fig. 7). This model is supported by studies of the acid-adapted stomach pathogen Helicobacter pylori, which maintains periplasmic pH through a CA-dependent mechanism (51, 52). Alternatively, CA may impact central carbon metabolism enzymes that rely on bicarbonate-CO2, such as PckA and other carboxylases, and modulate PhoPR through feedback associated with altered redox homeostasis or carbon metabolism. pH and the equilibrium of carbonic acid-bicarbonate-CO2 in water are physically linked, and in many biological systems, CO2 is indirectly sensed by monitoring pH (48). Notably, the PhoPR regulon is induced at a threshold of ∼pH 6.3 (5), the same pH at which the dissolved inorganic carbon equilibrium favors CO2 to dissolve in water. Therefore, it is tempting to speculate that PhoPR may function as a CO2 sensor that uses pH as a proxy for dissolved CO2.

Model linking ETZ, CA, and regulation of the PhoPR pathway. PhoPR is activated at a similar pH (∼6.3) as the dissolved inorganic carbon equilibrium that favors CO2 to dissolve in water. CA will interconvert CO2 + H2O to HCO3− + H+. Bicarbonate may be shuttled into the cytoplasm by a bicarbonate transporter (BT) to act in maintaining pH homeostasis or metabolism, while the proton produced in the reaction may promote the acidification of the extracellular environment surrounding PhoR, leading to induction of the PhoPR regulon. ETZ would inhibit this process by inhibiting CA and limiting the accumulation of protons in the pseudoperiplasm. This model is inspired by models from Sedlakova et al. (54) and Rasko and Sperandio (8).

Reporter strains are powerful tools for both the discovery and characterization of new antimicrobials. These strains are synthetic phenotypes and enable whole-cell screens to discover inhibitors of pathways that are otherwise inaccessible for high-throughput assays. Importantly, we show with the reporter strain used here that these strains can also be used as biomarkers to determine in vivo exposure of the bacterium to a newly discovered compound. In this manner, the reporter strain enables a rapid determination of whether the compound targets the pathway of interest in host tissues. Our study demonstrates the proof of concept that M. tuberculosis fluorescent reporter strains can be used for the discovery and in vivo characterization of inhibitors of M. tuberculosis two-component regulatory pathways. Many two-component signal transduction systems are required for M. tuberculosis pathogenesis (53); therefore, this approach may be applied to the discovery of other inhibitors that blind M. tuberculosis to environmental stimuli and attenuate virulence.

ACKNOWLEDGMENTS

We thank the New England Regional Center of Excellence (U54 AI057159) for providing the screening libraries and Su Chiang and Doug Flood for assistance in preparing the compound libraries for screening. The High-Performance Computing Cluster and iCER at Michigan State University provided computational support. The MSU RTSF provided technical support for the RNA-seq library preparation and sequencing. The MSU Center for Advanced Microscopy provided confocal microscopy support. We thank Kyle Rohde, Garry Coulson, Kathy Meek, Martha Mulks, and members of the Abramovitch lab for critical reading of the manuscript.

The research reported in this study from the Champion lab was supported by funding from the Great Lakes Regional Center of Excellence (GLRCE) (grant U54AI057153) and by the National Institute of Allergies and Infectious Diseases of the National Institutes of Health (NIH-NIAID) under award R01AI106872 to P.A.D.C. F.M.M. is supported by a postdoctoral fellowship from the Eck Institute for Global Health at the University of Notre Dame. Research reported in this study from the Abramovitch lab was supported by start-up funding from Michigan State University and AgBioResearch and grants from the NIH-NIAID (R21AI105867), the GLRCE (U54AI057153), the Michigan Economic Development Corporation (AGR2014-00199), and the Jean P. Schultz Endowed Biomedical Research Fund at the MSU College of Human Medicine.

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